Anti-microbial device and method for its manufacture

ABSTRACT

An antimicrobial medical device that includes a substrate having a metal surface that is made from a metal or metal alloy that may include stainless steel, cobalt, and titanium. Disposed on the metal surface is a first antimicrobial oxide layer that includes an antimicrobial metal that may include silver, copper, and zinc, and combinations thereof. The atoms of antimicrobial metal in the first antimicrobial oxide layer are of a first concentration. The first antimicrobial oxide layer is positioned in a direction opposite that of the metal surface. The device further includes a second antimicrobial oxide layer that includes an antimicrobial metal that may be silver, copper, and zinc, and combinations thereof. The atoms of the antimicrobial metal present in the second antimicrobial oxide layer are of a second concentration. The first concentration and the second concentration are not equal. Methods for making the antimicrobial medical device are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/776,980 filed on Jan. 30, 2020, which is a continuation of U.S.application Ser. No. 15/276,048 filed on Sep. 26, 2016, which is acontinuation of PCT Application No. PCT/US2015/022743 filed on Mar. 26,2015 which claims priority to U.S. Provisional Application No.61/970,501, filed on Mar. 26, 2014, each of which is herein incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to a medical device havingantimicrobial properties, and to a method of fabricating the medicaldevice.

BACKGROUND OF THE INVENTION

Infection is one of the most serious complications for medical devices,including implants. Efforts to rectify this problem have includedsurface-treatment with coatings that prevent bacterial adhesion, butsuch coatings typically are of limited effectiveness.

Surgical Site Infections (SSIs) involving medical devices (e.g.,orthopedic implants) are a well-known, widespread and severe problemleading to significant patient morbidity and mortality. Medical devicesoften serve as a nidus for bacterial colonization and biofilms thattrigger the formation of fibrous tissue around infected devices insteadof bone. This scenario further complicates patient outcomes by degradingbone and decreasing the device fixation required to stabilize thesegment (which is often the primary objective of the original surgery).Yet, the need to maintain the stability of the implant-bone interfacemakes leaving the device in place and attempting to treat the infectionwith, e.g., irrigation, debridement(s) and/or antibiotics the standardof care for many procedures, such as common spinal fusions.

Thus, a need exists for technology that addresses the problems ofcolonizing bacteria (preventing biofilm formation), and that, in somecases, also allows for the simultaneous and expeditious formation of astrong bone-to-implant interface that achieves construct stability.

While certain aspects of conventional technologies have been discussedto facilitate disclosure of the invention, Applicant in no way disclaimsthese technical aspects, and it is contemplated that the claimedinvention may encompass one or more of the conventional technicalaspects discussed herein.

In this specification, where a document, act or item of knowledge isreferred to or discussed, this reference or discussion is not anadmission that the document, act or item of knowledge or any combinationthereof was, at the priority date, publicly available, known to thepublic, part of common general knowledge, or otherwise constitutes priorart under the applicable statutory provisions; or is known to berelevant to an attempt to solve any problem with which thisspecification is concerned.

SUMMARY OF THE INVENTION

Briefly, the present invention satisfies the need for an antimicrobialmedical device and method of making the same that address the problem ofcolonizing bacteria. Some embodiments of the invention also allow forcontrolled antimicrobial release, and/or for the simultaneous andexpeditious formation of a strong bone-to-implant interface thatachieves construct stability.

In a first aspect, the invention provides an antimicrobial medicaldevice comprising:

-   -   a substrate comprising a metal surface, said metal surface        comprising atoms of at least one of a metal or metal alloy        comprising one or more of stainless steel, cobalt, and titanium;    -   on the metal surface, a first antimicrobial oxide layer        comprising atoms of the metal or metal alloy and atoms of an        antimicrobial metal selected from the group consisting of        silver, copper, and zinc, and combinations thereof, the atoms of        antimicrobial metal being present in the first antimicrobial        oxide layer in a first concentration; and    -   on the first antimicrobial oxide layer, positioned in a        direction opposite that of the metal surface, a second        antimicrobial oxide layer comprising atoms of an antimicrobial        metal selected from the group consisting of silver, copper, and        zinc, and combinations thereof, the atoms of antimicrobial metal        being present in the second antimicrobial oxide layer in a        second concentration,

wherein the first concentration is not equal to the secondconcentration.

In a second aspect, the invention provides a method for making anantimicrobial medical device, said method comprising:

-   -   providing a substrate comprising a metal surface, said metal        surface comprising atoms of at least one of a metal or metal        alloy comprising one or more of stainless steel, cobalt, and        titanium;    -   forming, on the metal surface, a first antimicrobial oxide layer        comprising atoms of the metal or metal alloy and atoms of an        antimicrobial metal selected from the group consisting of        silver, copper, and zinc, and combinations thereof, the atoms of        antimicrobial metal being present in the first antimicrobial        oxide layer in a first concentration; and    -   forming, on the first antimicrobial oxide layer, positioned in a        direction opposite that of the metal surface, a second        antimicrobial oxide layer comprising atoms of an antimicrobial        metal selected from the group consisting of silver, copper, and        zinc, and combinations thereof, the atoms of antimicrobial metal        being present in the second antimicrobial oxide layer in a        second concentration,

wherein the first concentration is not equal to the secondconcentration.

The present invention may address one or more of the problems anddeficiencies of the art discussed above. However, it is contemplatedthat the invention may prove useful in addressing other problems anddeficiencies in a number of technical areas. Therefore, the claimedinvention should not necessarily be construed as limited to addressingany of the particular problems or deficiencies discussed herein.

Certain embodiments of the presently-disclosed antimicrobial medicaldevices and methods for making the same have several features, no singleone of which is solely responsible for their desirable attributes.Without limiting the scope of these devices and methods as defined bythe claims that follow, their more prominent features will now bediscussed briefly. After considering this discussion, and particularlyafter reading the section of this specification entitled “DetailedDescription of the Invention,” one will understand how the features ofthe various embodiments disclosed herein provide a number of advantagesover the current state of the art. These advantages may include, withoutlimitation: providing devices that utilize a multi-layer antimicrobialloading that affectively addresses the problem of infection, as itoccurs in relation to medical devices, including over prolonged periodsof time; providing microbicidal technology to reduce colonizingbacteria; providing devices that are osteoinductive even in the presenceof bacteria; and providing devices that rapidly achieve fixation of bonesegment to stabilize the bone-device (e.g., bone-implant structure).Embodiments of the inventive antimicrobial medical devices and methodsof forming the same have widespread clinical relevance andapplicability, including, but not limited to spine, trauma, dental, andother applications.

These and other features and advantages of this invention will becomeapparent from the following detailed description of the various aspectsof the invention taken in conjunction with the appended claims and theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will hereinafter be described in conjunction withthe following drawing figures, wherein like numerals denote likeelements, and:

FIGS. 1A and 1B are schematics depicting the first and secondantimicrobial oxide layers on metal surfaces according to certainembodiments of the invention. FIG. 1C is a comparative prior artschematic depicting alternatively loaded nanoparticles absorbed withAg+, which are loaded to crystalline anatase TiO₂ not in accordance withthe present invention;

FIG. 2 depicts an embodiment of the inventive antimicrobial medicaldevice;

FIG. 3 is an image of a histological section of titanium hip step inaccordance with an embodiment of the invention with 3D porous scaffold;

FIG. 4A depicts pedicle screws in accordance with an embodiment of thepresent invention. FIG. 4B depicts comparative prior art pedicle screwsnot in accordance with an embodiment of the present invention;

FIG. 5 provides a chart showing data for S. aureus colony forming unitson various Ti rods inserted into the skin of guinea pigs in accordancewith controls and techniques used according to embodiments of theinvention;

FIG. 6 is a bar-graph showing standard human fibroblast cytotoxicity ofmatrices treated with antimicrobial agents commensurate with embodimentsof the present invention;

FIGS. 7A and 7B show the effect of antimicrobial agents used inembodiments of the present invention on inhibition of Staphylococcusepidermidis and Pseudomonas aeruginosa growth;

FIGS. 8A-C provide SEM and EDS image data relating to structuralfeatures and the chemical makeup of a nanofunctionalized deviceaccording to methods discussed herein, having a first antimicrobialoxide layer;

FIG. 9 provides line graphs that demonstrate the effects of differentanodization and soaking regimens on the amount and duration ofantimicrobial release from a treated matrix;

FIGS. 10A and 10B are photomicrographs that show S. aureus growth onanodized Ti-6Al-4V in the absence of antimicrobial treatment (control)(FIG. 10A) and on anodized Ti-6Al-4V that has been soaked forapproximately 30 minutes in an [Ag(NH₃)₂]NO₃ solution (FIG. 10B);

FIG. 11 is a line graph showing durations of bactericidal effect ofdevice matrices treated with antimicrobial oxide layers described hereindepending on the concentration of bactericidal solution in which theyhad been soaked during treatment;

FIG. 12 is a line graph showing durations of bactericidal effect ofdevice matrices treated with antimicrobial oxide layers described hereindepending on the concentration of bactericidal solution in which theyhad been soaked during treatment, upon continual presentation with abacterial challenge;

FIG. 13 is a bar-graph depicting bone growth on device matrices treatedwith antimicrobial agent;

FIG. 14 is an SEM image of anodized titanium alloy Ti-6Al-4V;

FIG. 15 is a line graph showing results of Ti-6Al-4V anodized underdifferent conditions;

FIG. 16 shows an SEM image of titanium alloy Ti-6Al-4V that includes aceramic layer loaded with antimicrobial silver ions;

FIG. 17 is graph of the spectrum of surface chemistry of an anodizedtitanium alloy surface coated with calcium phosphate; and

FIG. 18 is a line-graph showing bacterial growth in a culture wellcontaining a Ti-6Al-4V sample (control) and a silver loaded calciumphosphate coated anodized Ti-6Al-4V disc.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention and certain features, advantages, anddetails thereof, are explained more fully below with reference to thenon-limiting embodiments illustrated in the accompanying drawings.Descriptions of well-known materials, fabrication tools, processingtechniques, etc., are omitted so as to not unnecessarily obscure theinvention in detail. It should be understood, however, that the detaileddescription and the specific examples, while indicating embodiments ofthe invention, are given by way of illustration only, and are not by wayof limitation. Various substitutions, modifications, additions and/orarrangements within the spirit and/or scope of the underlying inventiveconcepts will be apparent to those skilled in the art from thisdisclosure.

Reference is made below to the drawings, which are not necessarily drawnto scale for ease of understanding, wherein the same reference numeralsretain their designation and meaning for the same or like elementsthroughout the various drawings.

In a first aspect, the invention provides an antimicrobial medicaldevice comprising:

-   -   a substrate comprising a metal surface, said metal surface        comprising atoms of at least one of a metal or metal alloy        comprising one or more of stainless steel, cobalt, and titanium;    -   on the metal surface, a first antimicrobial oxide layer        comprising atoms of the metal or metal alloy and atoms of an        antimicrobial metal selected from the group consisting of        silver, copper, and zinc, and combinations thereof, the atoms of        antimicrobial metal being present in the first antimicrobial        oxide layer in a first concentration; and    -   on the first antimicrobial oxide layer, positioned in a        direction opposite that of the metal surface, a second        antimicrobial oxide layer comprising atoms of an antimicrobial        metal selected from the group consisting of silver, copper, and        zinc, and combinations thereof, the atoms of antimicrobial metal        being present in the second antimicrobial oxide layer in a        second concentration,

wherein the first concentration is not equal to the secondconcentration.

As used herein, when an element (e.g., a layer) is referred to as being“on” (e.g., deposited on, formed on, disposed on, etc.) or “over”another element, it can be directly on the other element or interveningelements may also be present. In contrast, when an element is referredto as being “directly” on or over another element, there are nointervening elements present.

As used herein, the term “medical device” refers to any type ofdevice/appliance that is totally or partly introduced, surgically ormedically, into a patient's body and which may remain there after aprocedure or may be removed during treatment.

In some embodiments, the inventive antimicrobial medical device is adevice used for spine, trauma, or dental applications. In someembodiments, the antimicrobial medical device is a medical implant. Forexample, in certain embodiments, the medical device is an implantselected from an orthopedic implant and a neurosurgical implant.

Embodiments of antimicrobial medical devices according to the inventionhave microbicidal properties, due to the inclusion of at least the firstand second antimicrobial oxide layers. As discussed in greater detailbelow, it has been found that the multilayer antimicrobial loadingapproach used in the invention provides embodiments that are effectivein reducing colonizing bacteria, while simultaneously offeringosteoinductive properties that allow for rapid bone growth despite thepresence of bacteria. This is particularly advantageous in view of thefact that often, when a medical device is used, the device serves,within a patient, as a nidus for bacterial colonization and biofilmsthat trigger the formation of fibrous tissue around infected devicesinstead of bone, thereby resulting not only in infection, but also inconditions that degrade bone and prevent proper device fixation withinthe patient. Accordingly, embodiments of the inventive antimicrobialmedical device allow for improved patient outcomes via the reduction orprevention of infection, and by allowing proper device fixation andstabilization within the patient.

The inventive antimicrobial metal device includes a substrate, whichcomprises a metal surface. The metal surface comprises atoms of at leastone of a metal or metal alloy, the metal or metal alloy comprising oneor more of stainless steel, cobalt, and titanium. Accordingly, the metalsurface includes atoms from at least one of stainless steel, cobalt, andtitanium.

In some embodiments, the metal surface comprises titanium. For example,in some embodiments, the metal surface is fabricated of commerciallypure (CP) titanium. In some embodiments, the metal surface comprises ametal alloy. In particular embodiments, the metal alloy comprises one ormore of stainless steel, a titanium alloy (e.g., Ti6Al4V or nitinol),and a cobalt-chrome alloy.

In some embodiments, the metal surface is fabricated of, or consistsessentially of, CP titanium, stainless steel, a titanium alloy, or acobalt-chrome alloy.

In a particular embodiment, the metal surface is Ti6Al4V.

In some embodiments, the metal surface is nonporous, whereas in otherembodiments, the metal surface is porous.

The inventive medical device includes, disposed on the metal surface, afirst antimicrobial oxide layer comprising atoms of the metal or metalalloy and atoms of an antimicrobial metal selected from the groupconsisting of silver, copper, and zinc, and combinations thereof.

In various embodiments, the atoms of the antimicrobial metal are metalions (e.g., silver, copper, or zinc ions) that bind ionically to anoxidized surface or other portion of the medical device. If desired, insome embodiments, the metal ion may be subsequently reduced.

The ionic binding of the antimicrobial metal to an oxidized surfaceresults in the formation of a mixed oxide. For such embodiments, in thecase of the first antimicrobial oxide layer, a mixed oxide may be formedbetween the oxygen atom of an oxidized device surface or layer/structure(e.g., the oxidized metal surface) and an antimicrobial metal atom/ion.For example, in some embodiments, the first antimicrobial oxide layercomprises oxidized titanium. In non-limiting embodiments, said oxidizedtitanium may be naturally oxidized titanium, heat oxidized titanium,electrochemically treated titanium, etched titanium, or nano-anodizedtitanium. The oxidized titanium acts as a reservoir for ionicantimicrobial agents, which can bind ionically to the oxygen in thetitanium oxide, thereby forming a mixed oxide that comprises oxygenbound to the atom of the oxidized metal surface (e.g., Ti), and to theantimicrobial atom (e.g., ionic silver). For example, in someembodiments, ionic silver, e.g., from an aqueous [Ag(NH₃)₂]NO₃ solution,binds ionically to, and is stored on an oxidized titanium-comprisingsurface via the mechanism below:Ti—O⁻+[Ag(NH₃)₂]⁺→Ti—O—[Ag(NH₃)₂]

Persons having ordinary skill in the art will appreciate that the abovemechanism also supports binding to other metal oxides, and would beequally applicable using other cations comprising antimicrobial atoms(e.g., cations comprising silver, copper, and/or zinc).

In some embodiments, the first antimicrobial oxide layer comprises atomsof titanium or chromium (i.e., comprises atoms of at least one oftitanium and chromium).

In some embodiments, the first antimicrobial oxide layer comprises, asthe atoms of the antimicrobial metal, at least one of silver ions,copper ions, and zinc ions.

In particular embodiments, the first antimicrobial oxide layer comprisessilver atoms.

The atoms of the antimicrobial metal are present in the firstantimicrobial oxide layer in a first concentration. Examples ofacceptable methods for antimicrobial loading are discussed below.Persons having ordinary skill in the art will understand that thedesired concentration of antimicrobial atoms in the antimicrobial oxidelayers may vary depending on the intended device and its application.Nonetheless, in some non-limiting embodiments, the first concentrationranges from 0.5 to 60 μg of antimicrobial atoms per cm² (e.g., 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, or 60 μg/cm²), including any and all ranges and subrangestherein (e.g., 0.5 to 25 μg/cm²).

In some embodiments, the first antimicrobial oxide layer does notcomprise silicon.

In some embodiments, the first antimicrobial oxide layer does notcomprise a ceramic material.

In some embodiments, the first antimicrobial oxide layer does notcomprise, and is not in direct contact with, zeolite.

In some embodiments, the first antimicrobial oxide layer is nonporous,whereas in other embodiments, the first antimicrobial oxide layer isporous.

The inventive antimicrobial medical device also includes, on the firstantimicrobial oxide layer, positioned in a direction opposite that ofthe metal surface, a second antimicrobial oxide layer. The secondantimicrobial oxide layer also comprises atoms of an antimicrobial metalselected from the group consisting of silver, copper, and zinc, andcombinations thereof, which are present in the second antimicrobialoxide layer in a second concentration.

In various embodiments, the atoms of the antimicrobial metal are metalions (e.g., silver, copper, or zinc ions) that bind ionically to anoxidized surface or other portion of the medical device. If desired, insome embodiments, the metal ion may be subsequently reduced.

As in certain embodiments of the first antimicrobial oxide layer, in thecase of certain embodiments of the second antimicrobial oxide layer, theionic binding of the antimicrobial metal to an oxidized surface resultsin the formation of a mixed oxide. For such embodiments, a mixed oxidemay be formed between the oxygen atom of an oxidized device surface orlayer/structure and an antimicrobial metal atom/ion. For example, insome embodiments, the second antimicrobial oxide layer comprisesoxidized titanium. In non-limiting embodiments, said oxidized titaniummay be naturally oxidized titanium, heat oxidized titanium,electrochemically treated titanium, etched titanium, or nano-anodizedtitanium (e.g., in fluorine-containing electrolyte). The oxidizedtitanium acts as a reservoir for ionic antimicrobial agents, which canbind ionically to the oxygen in the titanium oxide, thereby forming amixed oxide that comprises oxygen bound to the atom of the oxidizedmetal surface (e.g., Ti), and to the antimicrobial atom (e.g., ionicsilver).

In some embodiments, the second antimicrobial oxide layer comprisesatoms of titanium or chromium (i.e., comprises atoms of at least one oftitanium and chromium).

In some embodiments, the second antimicrobial oxide layer comprises, asthe atoms of the antimicrobial metal, at least one of silver ions,copper ions, and zinc ions.

In some embodiments, the second antimicrobial oxide layer comprises amixed oxide that contains atoms of at least one of stainless steel,cobalt, and titanium.

In particular embodiments, the second antimicrobial oxide layercomprises silver atoms.

The atoms of the antimicrobial metal are present in the secondantimicrobial oxide layer in a second concentration, which is differentfrom the first concentration. In some non-limiting embodiments, thesecond concentration ranges from 0.5 to 60 μg of antimicrobial atoms percm² (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, or 60 μg/cm²), including any and all rangesand subranges therein (e.g., 5 to 25 μg/cm²), with the proviso that thesecond concentration does not equal the first concentration.

In some embodiments, the first and or second oxide layer has a thicknessof about 100 nm to 3 μm (e.g., 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730,740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1500,2000, 2500, or 3000 nm), including any and all ranges and subrangestherein.

In some embodiments, the second concentration is less than the firstconcentration.

In other embodiments, the second concentration is greater than the firstconcentration.

In some embodiments, there is at least a 10% difference between thefirst concentration and the second concentration

$\left( {{i.e.},\ {{\frac{\begin{matrix}{\left( {{first}\mspace{14mu}{concentration}} \right) -} \\\left( {{second}\mspace{14mu}{concentration}} \right)\end{matrix}}{{first}\mspace{14mu}{concentration}} \times 100\%}\  \geq {10\%}}} \right).$

The concentration of the atoms of antimicrobial metal in the first andsecond antimicrobial oxide layers can be carefully controlled and isbelow concentrations that would cause dangerous toxicological sideeffects. In various embodiments, the antimicrobial release profile(e.g., of silver ions, or of whatever other antimicrobial atoms areused) is controlled by different binding and/or loading (e.g.,antimicrobial layering) strategies.

In some embodiments, the second antimicrobial oxide layer does notcomprise silicon.

In some embodiments, the second antimicrobial oxide layer does notcomprise a ceramic material.

In some embodiments, the second antimicrobial oxide layer does notcomprise, and is not in direct contact with, zeolite.

In some embodiments, the second antimicrobial oxide layer is nonporous,whereas in other embodiments, the second antimicrobial oxide layer isporous.

In some embodiments, the second antimicrobial oxide layer is disposeddirectly on the first antimicrobial oxide layer (i.e., the layers are indirect contact with one another).

In various embodiments, the layering (i.e., the at least first andsecond antimicrobial oxide layers) provides an antimicrobial medicaldevice that is configured to allow for controllable sustained release ofantimicrobial agent.

In some embodiments, for example, in embodiment 100 of FIG. 1A, thefirst antimicrobial oxide layer 8 and second antimicrobial oxide layer10 are distinct layers separated from one another by at least anintermediate layer 6, which may be, e.g., porous by anodization, flat bydeposition, etc. (i.e., the layers are not in direct contact with oneanother). In some embodiments, the intermediate layer 6 is fabricatedfrom the same material as the metal surface. In other embodiments, theintermediate layer is fabricated from a different material than themetal surface. FIG. 1A includes an optional additional antimicrobialoxide layer 12, which is separated from the second antimicrobial oxidelayer 10 by a second intermediate layer 6. In the depicted embodiment100, the first antimicrobial oxide layer 8 is formed ontitanium-comprising substrate 2.

In some embodiments, the intermediate layer comprises atoms of at leastone of a metal or metal alloy comprising one or more of stainless steel,cobalt, and titanium.

In some embodiments, the intermediate layer does not comprise anantimicrobial metal selected from the group consisting of silver,copper, and zinc, and combinations thereof.

In some embodiments of the inventive antimicrobial medical device, thedevice comprises a plurality of nanostructures.

In some embodiments, the first and/or second antimicrobial oxide layersare contained within the plurality of nanostructures.

In some embodiments the nanostructures are disposed directly on themetal surface. In some embodiments, the nanostructures are disposed on alayer that is disposed on the metal surface.

While the nanostructures may be of any known geometry, in particularembodiments, the nanostructures are nanotubes.

In some embodiments, the nanostructures comprise titanium dioxide.

In some embodiments, the nanostructures are amorphous non-crystallinenanostructures. For example, in some embodiments, the nanostructurescomprise amorphous titanium dioxide (versus crystalline titanium dioxidesuch as, e.g., anatase). FIG. 1B depicts such an embodiment 200 whereinthe first antimicrobial oxide layer 8 and second antimicrobial oxidelayer 10 are contained within amorphous titanium dioxide nanotubes 4that are formed on titanium-comprising substrate 2. The first and secondantimicrobial oxide layers 8 and 10 are formed by loading ions (e.g.,Ag+) directly to amorphous titanium dioxide nanotubes in a solution ofsilver ammonia nitrate. It is noted that the multi-layering embodimentsof the present invention, such as that of FIG. 1B differ from, forexample, embodiments described in EP 2495356, which do not use such alayering technique, and wherein, as shown in embodiment 300 of FIG. 1C,silver ions 14 are loaded to titanium dioxide nanoparticles 16, whichare then loaded into annealed crystalline nanotubes 5.

In some embodiments, the inventive antimicrobial medical device furthercomprises a ceramic layer (e.g., calcium phosphate). In someembodiments, the ceramic layer comprises atoms of an antimicrobial metalselected from the group consisting of silver, copper, and zinc, andcombinations thereof.

In some embodiments, the inventive antimicrobial medical device does notcomprise a ceramic material disposed on (directly, or indirectly) themetal surface.

In some embodiments of the inventive antimicrobial medical device, thesubstrate comprises a body of a device, such as an implant. For example,in some embodiments, the metal surface is disposed on a substrate thatcomprises a metallic, ceramic, stainless steel, polymeric (e.g.,polyether-ether-ketone (PEEK)), or other implant. In some embodiments,the substrate is a three-dimensional structure.

In some embodiments, the inventive antimicrobial medical device has atleast one of pico, micron, sub-micron, nano or meso-scale surfacefeatures, or a smooth surface (e.g., a smooth but porous surface), thatfacilitates tissue attachment and growth at an interface between themedical device and tissue or bone.

In a second aspect, the invention provides a method for making anantimicrobial medical device, said method comprising:

-   -   providing a substrate comprising a metal surface, said metal        surface comprising atoms of at least one of a metal or metal        alloy comprising one or more of stainless steel, cobalt, and        titanium;    -   forming, on the metal surface, a first antimicrobial oxide layer        comprising atoms of the metal or metal alloy and atoms of an        antimicrobial metal selected from the group consisting of        silver, copper, and zinc, and combinations thereof, the atoms of        antimicrobial metal being present in the first antimicrobial        oxide layer in a first concentration; and    -   forming, on the first antimicrobial oxide layer, positioned in a        direction opposite that of the metal surface, a second        antimicrobial oxide layer comprising atoms of an antimicrobial        metal selected from the group consisting of silver, copper, and        zinc, and combinations thereof, the atoms of antimicrobial metal        being present in the second antimicrobial oxide layer in a        second concentration,

wherein the first concentration is not equal to the secondconcentration.

The inventive methods of making an antimicrobial medical device may beused to fabricate the inventive antimicrobial medical device accordingto embodiments of the first aspect of the invention.

In some embodiments, at least one of forming the first antimicrobialoxide layer and forming the second antimicrobial oxide layer comprisesexposing at least a portion of the device to a solution comprising anantimicrobial metal selected from the group consisting of silver,copper, and zinc, and combinations thereof, or to an antimicrobial metalion thereof. Generally, the solution is a solution that comprises asolvent (e.g., water) and a soluble silver, copper, or zinc salt.

In some embodiments, “exposing at least a portion of the device” to thesolution comprises exposing the metal surface and/or a portion of thedevice disposed, either directly or indirectly, on the metal surface, tothe solution. For example, in some embodiments, the exposing comprisesexposing nanostructures or a portion thereof to the solution. In someembodiments, both forming the first antimicrobial oxide layer andforming the second antimicrobial oxide layer comprises this exposingprocess.

In some embodiments, the solution comprises silver ions, copper ions,and/or zinc ions. In particular embodiments, the solution comprisessilver or silver ions.

In some embodiments, the solution is made by dissolving a silver,copper, or zinc salt in solvent. In some embodiments, the solvent iswater. In some embodiments, the solvent is water and the salt is asilver salt. In some embodiments, the silver salt is [Ag(NH₃)₂]NO₃.

In some embodiments, the first antimicrobial oxide layer is formeddirectly on the metal surface (i.e., in direct contact with the metalsurface).

While oxide layers form naturally on various metal and metal alloysurfaces, there are also many known methods for forming (includingenhancing) oxide layers. For the present invention, the first and secondantimicrobial oxide layers may be formed by reacting a precursor oxidelayer (whether a natural oxide layer or an otherwise formed or enhancedoxide layer) with antimicrobial atoms. While any art acceptable methodsmay be employed to form the oxide layer, including the precursor oxidelayer, in some embodiments, forming the first and/or secondantimicrobial oxide layer comprises mechanical roughening, heattreatment, acid etching, and/or electrochemical oxidization. In someembodiments, forming the first and/or second antimicrobial oxide layercomprises anodizing.

In some embodiments, forming the first antimicrobial oxide layercomprises heat treating the metal surface, acid etching the metalsurface, or electrochemically oxidizing the metal surface. In someembodiments, forming the first antimicrobial oxide layer comprisesanodizing the metal surface.

In some embodiments, forming the second antimicrobial oxide layercomprises depositing a metal (e.g., a metal layer or structure), on thefirst antimicrobial oxide layer, and forming the second antimicrobialoxide layer directly on the metal, either via reaction with a naturaloxide on the metal, or by first applying an oxidizing treatment to asurface of the metal. While the metal layer or structure may be formedusing any art-acceptable method, in some embodiments, it is spray-coatedon the substrate (e.g., onto the metal surface or first antimicrobialoxide layer), formed using physical vapor deposition (PVD), for formingby anodizing.

In some embodiments, the inventive method comprises forming on the metalsurface a plurality of nanostructures, which may optionally contain thefirst and second antimicrobial oxide layers. In some embodiments, theplurality of nanostructures are formed by anodizing. As will beappreciated by those skilled in the art, by varying anodizationparameters, different structures with different sizes (nano or, in someembodiments, micron), morphologies, surface energies and otherproperties may be created.

In some embodiments, the nanostructures are nanotubes. In someembodiments, the nanotubes have an average diameter of less than orequal to 100 nm. In some embodiments, the nanotubes have an averagediameter of 5 to 100 nm (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nm), includingany and all ranges and subranges therein.

Nanotube length can easily be modified depending on the intended medicaldevice and its application. While nanotubes of any desired length areencompassed by the invention, in some embodiments, the nanotubes have anaverage length ranging from 15 nm to several microns. In someembodiments, the nanotubes have an average length of 20 nm to 1,000 nm(e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or1000 nm), including all ranges and subranges therein.

In some embodiments, the inventive method comprises formingnanostructures on the metal surface (e.g., by anodizing), then formingthe first antimicrobial oxide layer, then, after forming the firstantimicrobial oxide layer, further anodizing to continue to form thenanostructures, and subsequently forming the second antimicrobial oxidelayer, which may be comprised within the nanostructures. By stopping andrestarting the formation of nanostructures e.g., nanotubes) byanodization, the antimicrobial metal in solution can be modified toincrease or decrease the amount or type of antimicrobial agentincorporated into the portion of the device being loaded withantimicrobial material. This allows for customized elution profiles tobe created. In other embodiments, this effect may also be achieved bysubjecting different portion(s) of the antimicrobial medical device todifferent loading conditions (e.g., a first part of the device may besoaked in antimicrobial solution, followed by a separate soak of adifferent part of the device, or of only a portion of the first part).

In some embodiments, the inventive method comprises a method of makingan antimicrobial medical device that comprises a Ti-6Al-4V metalsurface, wherein the first and second antimicrobial oxide layers areformed on titanium dioxide nanostructures. For example, in particularembodiments, substrates comprising anodized Ti-6Al-4V metal surfaces arethoroughly cleaned and dried to remove any surface contamination. Duringanodization, the anodized titanium substrates serve as anode in anelectrochemical cell. An electrolyte is used, which can vary incomposition and concentration. For example, in some embodiments, dilutehydrofluoric acid is used as an electrolyte. In some embodiments, thedilute HF electrolyte ranges from 0.25% to 1.5% HF, including any andall ranges and subranges therein.

During anodization, voltage is used as is known in the art. For example,in some embodiments, the voltage can vary from 2 to 45 V (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, or 45 V), including any and all ranges and subranges therein(e.g., from 5 to 20 V). Duration is controlled depending on the desirednanostructures to be form. For example, in some embodiments, theanodizing treatment ranges from 10 seconds to 20 minutes. Theas-oxidized titanium may be soaked in an aqueous solution of[Ag(NH₃)₂]NO₃. In some embodiments, to prepare the solution, droplets ofammonia hydroxide are added into 20 wt % silver nitrate untilprecipitates all dissolve. The stock solution is further diluted toprepare 0.001M to 0.1 M working solution.

In some embodiments, soaking (in antimicrobial metal solution) isperformed to form one or more antimicrobial oxide layers on thenanostructures. In some embodiments, the soaking process lasts from,e.g., 10 minutes to 24 hours. The soaking process may be one-time(single dose) or repeated (multiple dose). In some multi-doseembodiments, the substrates is rinsed with water thoroughly, completelydried in the air, and re-soaked in silver containing solution. In someembodiments, the soaking process may occur after the surface is fullyoxidized. In this case, silver ions bind to layers of surface oxides andresult in a relatively quick release profile.

In various embodiments, the soaking process may be performed repeatedlyat different stages of oxidation process (heat treatment, anodization,or mixed). For example, a substrate may be first anodized for, e.g., 10seconds, to initiate nanostructure (e.g., nanotube) formation. Then itmay be soaked in silver solution to adsorb ionic silver. A heattreatment may be used to fuse the silver ions into the underlyingsurface but not change the nanotube morphology. Subsequently, thesubstrate may be repeatedly (once or more) anodized to grow the nanotubestructures, soaked, and optionally heated to produce multipleantimicrobial layers within the surface structure. In some embodiments,the superficial surface of the substrate is modified with silver ionswithout further heating.

In some embodiments, the inventive method comprises forming one or moreadditional antimicrobial oxide layers in addition to the first andsecond antimicrobial oxide layers. Any additional oxide layer(s) may becomprised of the same, or a different material than the first and secondantimicrobial oxide layer. In some embodiments, at least one of one ormore additional antimicrobial oxide layers present is contained withinnanostructures that are disposed on the metal surface.

In some embodiments, the inventive method comprises forming on theantimicrobial medical device (e.g., on the metal surface) a ceramiclayer (e.g., calcium phosphate). In some embodiments, the ceramic layercomprises an antimicrobial material. For example, in some embodiments,the ceramic layer comprises atoms of an antimicrobial metal selectedfrom the group consisting of silver, copper, and zinc, and combinationsthereof.

In some embodiments, the inventive method comprises forming, on thefirst antimicrobial oxide layer, in a direction opposite that of that ofthe metal surface, an intermediate layer. In some embodiments, theintermediate layer is formed between the first and second antimicrobialoxide layers.

While the intermediate layer may be formed using any art-acceptablemethod, in some embodiments, the intermediate layer is spray-coated onthe substrate (e.g., onto the metal surface or first antimicrobial oxidelayer), or is formed using physical vapor deposition (PVD).

In some embodiments, the inventive method comprises disposing the metalsurface of the antimicrobial medical device on a three-dimensionalstructure, such as an implant. As used in this context, the metalsurface would be considered to be disposed on the structure regardlessof whether the structure is first provided and the metal surface isformed thereon, or whether the metal surface is formed, and thestructure is thereafter formed thereunder. In some embodiments, themethod comprises forming the metal surface, and thereafter joining thethree-dimensional structure to the metal surface. For example, in someembodiments, the inventive method comprises injection molding athree-dimensional structure (e.g., the body of an implant) to the metalsurface. In some embodiments, the three-dimensional structure comprisesa metallic, ceramic, stainless steel, polymeric (e.g.,polyether-ether-ketone (PEEK)), or other material.

EXAMPLES

The invention will now be illustrated, but not limited, by reference tothe specific embodiments described in the following examples.

Example 1: Interbody Fusion Device (IFD) Having an AntimicrobialOsteoinductive Nanotube Surface

A sustained-release of bacteria-reducing ions was developed on anosteoinductive nanotube surface platform applied to a porous titaniumscaffold-PEEK hybrid implant substrate, thereby forming antimicrobialIFD 400 as shown in FIG. 2 . Specifically, the substrate was a poroustitanium metal surface (Ti6Al4V), with PEEK 20 injection molded directlyinto the porous scaffold, thus resulting in an IFD with a very strongattachment strength to PEEK. Silver ion-comprising antimicrobial oxidelayered titanium dioxide nanotubes (as depicted in FIG. 1B) were formedon the metal surface, thereby forming metal surface scaffold 18.Specifically, using a fluorine-containing electrolyte and low constantvoltages, the IFD is treated to possess nanotubular structures. Theanodization system is a two electrode circuit with titanium-containingpart serving as an anode and a high purity platinum sheet (Alfa Aesar)as a cathode. The two electrodes are connected to a DC power supply anda constant voltage is applied during the anodization. During processing,the anode and cathode are kept with a separation distance of about 1 cm,and submerged into an electrolyte solution composed of dilute HF (0.5 wt%). The anodization voltage is adjusted from 3 to 25 V to producenanotubes with increasing diameters. Next, a solution soaking method isused to allow silver ion exchange on the surface of the porous titaniumscaffold. To do that, as-anodized titanium was soaked in an aqueoussolution of [Ag(NH₃)₂]NO₃. To prepare the silver ammonia nitratesolution, droplets of ammonia hydroxide were added into 20 wt % silvernitrate until the precipitates all dissolved. The stock solution wasfurther diluted to prepare 0.001M to 0.1 M working solutions. Thesoaking process lasted from 10 min to 24 hours and was either a one-time(single dose) or repeated (multiple dose). To do this, the substrateswere rinsed with water thoroughly, completely dried in the air, andre-soaked in a silver containing solution. For the purpose of prolongedsilver ion release, we tried more advanced methods to load the silvercontent.

The IFD leaves an open porous scaffold (i.e., the nanotubes) on thebone-opposing surface. Importantly, bone typically needs to grow only300 microns into the antimicrobial osteo-integrative nanotube scaffoldto achieve stability. For conventional implants, on the other hand,trabecular bone must grow through a distance of 5 to 16 mm, a processthat typically takes 6 to 12 months or much longer (if ever), in thepresence of surgical site infections. Accordingly, this and otherembodiments of the inventive antimicrobial medical device are expectedto achieve bone to implant fixation up to 80% faster than the standardof care, despite the presence of a surgical site infection. The IFD isable to maintain a release of a minimum inhibition concentration (MIC)to kill common bacteria strains observed in spinal implant infection forup to 2 weeks and is also able to control the speed and amount ofreleased agents to minimize the toxicity of the ions, allowing thenanosurface morphology and surface energy advantages to increaseosseointegration.

The IFD 400 provides a bone ingrowth scaffold that is 60-70% porous withinterconnected pores that average 523 μm in diameter and would have veryhigh attachment strength between the titanium scaffold and PEEK. IFD 400is manufactured by diffusion bonding porous sheets together to create athree-dimensional porous structure. The porous sheets are typicallyetched with through holes to generate the porosity. After diffusionbonding, the scaffold may be milled, electric discharge machined,stamped, and/or formed to desired geometries. It can be diffusion bondedto titanium and cobalt chrome substrates and likewise, polymers such asPEEK are injection and/or compression molded into inserts to createpolymer implants with titanium ingrowth regions. The strength of themechanical bond between the PEEK and scaffold exceeds the 2900 psistrength requirement.

IFD 400 and other inventive embodiments are advantageous as compared toother substitute technologies such as plasma spray IFDs. Historically,plasma sprayed titanium has delaminated from PEEK, causing wear debrisconcerns whereas the novel injection molding approach provides verystrong attachment of the scaffold to PEEK. Additionally, the bestfixation strength possible for existing surfaces is determined by thelimits of bone on growth to the surface of the IFD whereas inventiveembodiments, including IFD 400, provide bone ingrowth into a scaffold.Literature on bone attachment strength to porous bone scaffolds, withoutfurther nanotube surface features and no infection suggests much higherattachment strength than simply rough surfaces. Trabecular metalimplants come with similar porous structures but lack the desirableradiolucency and modulus offered by PEEK substrate.

The inventive multilayered antimicrobial surfaces, such as the nanotubesurface on IFD 400, improve osseointegration to the implant surface.Optimized titanium nanotubular surface properties (including chemistry,morphology, wettability, etc.) can have significant effects on bone toimplant fixation and therefore fixation and stability of the treatedsegment. The anodization technique used to form the nanotubes on IFD 400can: (A) form a thin layer of titanium oxides on the surface which hasbeen proven to be favored by bone cells; (B) incorporate hydroxyl groupsso as to increase wettability to increase the adsorption of proteinsknown to decrease bacteria functions and increase bone cell functions;(C) create patterned nanostructures (specifically, nanotubes) uniformlyover the surface, with controllable

parameters (diameter, length, etc.) to direct bone cell functions; and(D) provide a good matrix for drug delivery, including the proposedionic antibacterial agents (e.g., Ag+). The inventive layered andoptionally nanostructured material constructs can be applied across allof divisions of orthopedics and neurosurgery that both treats infectionand accelerates osseointegration and segmental stabilization despiteSSI. Most importantly, this may all be accomplished without the use ofdrugs, since any drug has multiple effects in the body, not just thedesired effect. The inventive embodiments also incorporate and stage therelease of antimicrobial ions from the antimicrobial oxide layers on themetal surface so as to achieve rapid bone to implant fixation andsegmental stabilization in both the absence and presence of infection.

Example 2: 3D Titanium Porous Scaffold onto PEEK Substrate—Animal Study

By diffusion bonding CAD designed porous titanium sheets together, 3Dporous scaffolds with a thickness of around 1 mm are created. PEEK cagesare compression molded into inserts to create a composite IFD withtitanium ingrowth regions on both sides. The 3D scaffold is studied in adog tibia model (see FIG. 3 ) and it is found that the fraction ofavailable void space filled with bone and tissue after 12 weeks was0.754 (σ=0.093). This preliminary animal study shows the average boneingrowth for fiber titanium is reported to range from 0.23-0.38 in a 12week study using a canine THR model.

Example 3: Pedicle Screws with Nanotubes 30 nm in Diameter

FIG. 4A depicts anodized titanium pedicle screws with nanotubes 30 nm indiameter in sheep pedicles after 3 months. The screws are titaniumscrews that were anodized to form titanium dioxide nanotubes withantimicrobial oxide layers formed therein, as described in Example 1,above. FIG. 4B depicts titanium pedicle screws without any nanotubes andantimicrobial oxide layers formed therein. As can be seen by comparingFIGS. 4A and 4B, use of the inventive embodiment significantly reducedscrew loosening compared to conventional screws.

Example 4: Efficacy of Silver Loaded Nanotubular Titanium on ReducingBacteria Growth

Two full-thickness incisions, 0.5-cm apart were created on the skin ofguinea pigs. Various titanium wires (consisting of controls (nomodification) (“plain Ti” in FIG. 5 ), anodized to possess nanotubes(“Anodized 30 nm tubes” and “Anodized 70 nm tubes” in FIG. 5 ) andanodized to possess nanotubes having a first antimicrobial oxide layer(“Anodized 30 nm tubes with Ag and “Anodized 30 nm tubes with doubledose of Ag) were separately inserted into the wound site. For thebacteria challenge study group, the surgical sites (insertion and exitsite) in half of the animals receiving implants were randomly inoculatedwith a standard aliquot of 1×10⁶ S. aureus (ATCC, strain no. 29213).Dorsal skin sections containing the rods were harvested 7 days afterimplantation and biofilm analyzed for colony forming unit by sonicationat 50 Hz for 7 min followed by agar. Experiments were conducted intriplicate. FIG. 5 provides a chart that summarizes the results, whichevidence decreased S. aureus growth when using anodized titanium coatedwith Ag ions compared to anodized titanium alone or plain titanium. A⅕^(th) and a 5 log reduction was found when using anodized Ti alone andanodized Ti with Ag ions, respectively. In FIG. 5 , Data=mean+−SEM;N=3; * p<0.01 compared to plain titanium and ** p<0.01 compared toanodized titanium alone. Y axis is colony forming units×106. Ti wasanodized at 10 or 20 V and then soaked in 0.001M [Ag(NH3)2]NO3 for 30 or60 minutes (double dose).

Example 5: Inventive Embodiments have Acceptable Cytotoxicity

FIG. 6 is a bar-graph showing standard human fibroblast cytotoxicity ofmatrices treated with antimicrobial agents commensurate with embodimentsof the present invention. Anodized Ti-6Al-4V either was notsilver-loaded or was (i) anodized in 0.5% HF solution for 10 min at 20Vto form short nanotubes, (ii) anodized in 0.5% HF/16.7 H₂O/DMSO solutionfor 2 hours at 40 volts to form long nanotubes, and then silver-loadedby being soaked in 0.001M [Ag(NH₃)₂]NO₃ for 30 minutes. Extracts fromindividual samples after 24 h soaking were added into a pre-culturedfibroblast plate and the proliferation of human fibroblast was thenmeasured using standard MTT assay as shown on the Y-axis in arbitraryunits in FIG. 6 . As can be seen, human fibroblasts growth exposed toextracts from anodized Ti-6Al-4V soaked in 0.001M solution for 30minutes, with varied nanotube thickness, were equivalent to that onnon-silver-loaded surface, thus demonstrating acceptable cytotoxicityfor the inventive embodiments.

Example 6: Inventive Embodiments have Acceptable Cytotoxicity

FIGS. 7A and 7B show the effect of antimicrobial agents used inembodiments of the present invention (namely, silver ions) on inhibitionof Staphylococcus epidermidis and Pseudomonas aeruginosa growth. Forthis example, Ti-6Al-4V was anodized in an HF solution then soaked forapproximately 30 minutes in an 0.001M [Ag(NH₃)₂]NO₃ solution. 50 ul ofeach bacteria strain at a concentration of 2.5×10⁶ cfu/ml was inoculatedonto an agar plate S. with the silver-loaded sample on top of it. As seein FIGS. 7A (for Staphylococcus epidermidis) and 7B (for the Pseudomonasaeruginosa), after 24 hours an inhibition zone appeared for bothbacteria strains.

Example 7: Structural and Chemical Analysis of Anodized Ti-6Al-4VTreated with Silver Solution

FIGS. 8A-C provide SEM and EDS image data relating to structuralfeatures and the chemical makeup of a nanofunctionalized deviceaccording to methods discussed herein, having a first antimicrobialoxide layer. For this example, a Ti-6Al-4V metal surface was anodized ina 0.5% solution of HF for 10 minutes at 10 V. Following anodization, theobject was soaked in 0.001M [Ag(NH₃)₂]NO₃ for 30 minutes, then dried.The micro and nanoscale surface features of the device were then imagedby scanning electron microscopy (SEM), and the elemental composition ofthe surface determined by energy dispersive spectrometry (EDS).Microscopy and spectrometry were conducted according to standard methodswhich would be known to ordinarily skilled artisans. The images shown inFIGS. 8A and 8B are SEM images at 20K magnification and 150Kmagnification, respectively. The formation of nanotubular structures onthe matrix of the object can be seen at higher magnification (FIG. 8B).FIG. 8C provides an EDS spectrum that shows emissions characteristic ofelemental silver, indicating the adsorption of silver (wt % ranging from1 to 4%, i.e., of 100 grams of atoms on the surface, 1 to 4 would besilver) onto device so as to form at least the first antimicrobial oxidelayer. The highest peak, at approximately 4.5 keV, represents elementaltitanium.

Example 8: Effects of Different Anodization and Soaking Regimens

The line graphs of FIG. 9 demonstrate the effects of differentanodization and soaking regimens on the amount and duration ofantimicrobial release from a treated matrix. For these examples, discsof Ti-6Al-4V were anodized in an HF solution as described above, thensoaked in 0.01M [Ag(NH₃)₂]NO₃. Silver release from the discs was thenmeasured, as shown on the Y-axis, over a period of twelve (12) days, asshown on the X-axis. Graph A shows release behavior of a disc that wasanodized once then repeatedly soaked in 0.01M [Ag(NH₃)₂]NO₃. As can beseen, there was a burst of silver release on day 1 from a disc receivingthis treatment, and most of the silver had been released by day 4. GraphB, by comparison, shows release behavior of a disc that was,sequentially, anodized as described, soaked as described, againanodized, then again soaked, so as to form first and secondantimicrobial oxide layers. As can be seen, this repeated alternationbetween anodization and soaking resulted in more prolonged release ofsilver and less concentrated levels of release.

Example 9: Bactericidal Effect of Treating Matrices with SilverSolutions as Described Herein

FIGS. 10A and 10B are photomicrographs that show S. aureus growth onanodized Ti-6Al-4V in the absence of antimicrobial treatment (control)(FIG. 10A) and on anodized Ti-6Al-4V that has been soaked forapproximately 30 minutes in an [Ag(NH₃)₂]NO₃ solution (FIG. 10B). Forthis example, Ti-6Al-4V was anodized in an HF solution then soaked forapproximately 30 minutes in an [Ag(NH₃)₂]NO₃ solution (FIG. 10B) anddried as described above or not soaked in [Ag(NH₃)₂]NO₃ (FIG. 10A). S.aureus was then cultured on the surfaces. As see in FIG. 10B, after four(4) hours, few colonies survived on the surface that had been soaked in[Ag(NH₃)₂]NO₃, indicating the bactericidal effect of treating matriceswith solutions as described herein. On the other hand, FIG. 10A showsgrowth of significant colonies.

Example 10: Durations of Bactericidal Effect of Treated Devices withAntimicrobial Oxide Layers

The line-graph in FIG. 11 shows durations of bactericidal effect ofdevice matrices treated with antimicrobial oxide layers described hereindepending on the concentration of bactericidal solution in which theyhad been soaked during treatment. The Y-axis shows bacterial growth inarbitrary units, measured over several days, as shown on the X-axis. Forthis example, Ti-6Al-4V discs were anodized for 10 minutes in a 0.5% HFsolution, then ionic silver-loaded by incubation for 30 minutes in asolution containing the indicated concentrations of [Ag(NH₃)₂]NO₃. Thecontrol was not incubated in [Ag(NH₃)₂]NO₃. Discs were then placed inculture wells, then challenged once with 1 ml of Luria broth containing10⁷/ml S. aureus, as shown in FIG. 11 . Note that this concentration ofS. aureus is much higher than a more physiologically relevantconcentration such as approximately 10⁴/ml. As can be seen in FIG. 11 ,the control substrate permitted bacterial growth from day 1 while allsilver-loaded substrates inhibited bacterial growth up to three (3)days; growth in the lowest concentration sample was seen on day 4 due toexhausted silver release.

FIG. 12 is a line graph showing durations of bactericidal effect ofdevice matrices treated with antimicrobial oxide layers described hereindepending on the concentration of bactericidal solution in which theyhad been soaked during treatment, upon continual presentation with abacterial challenge. The Y-axis shows bacterial growth in arbitraryunits, measured over several days, as shown on the X-axis. For thisexample, Ti-6Al-4V discs were anodized for 10 minutes in a 0.5% HFsolution, then ionic silver-loaded by incubation for approximately 30minutes in a solution containing the indicated concentrations of[Ag(NH₃)₂]NO₃. The control was not incubated in [Ag(NH₃)₂]NO₃. Discswere then placed in culture wells, then challenged with 1 ml of Luriabroth containing 10⁷/ml S. aureus every day for four consecutive days,as shown in FIG. 12 . As can be seen in FIG. 12 , the control substratepermitted bacterial growth from day 1, while all silver-loadedsubstrates inhibited bacterial growth up to three (3) days or more evenafter repeated daily inoculation with fresh bacterial stock; growth inthe lower concentration samples began to be seen on day 3, and in thehighest concentration sample growth began to be seen on day 4, due toexhausted silver release.

Example 11: Bone Growth on Device Matrices Treated with Silver

FIG. 13 is a bar-graph depicting bone growth on device matrices treatedwith antimicrobial agent. For this example, anodized Ti-6Al-4V eitherwas not silver-loaded or was silver-loaded by being soaked in 0.001M[Ag(NH₃)₂]NO₃. Soaking duration was for either 30 minutes or 60 minutes.Proliferation of human osteoblasts on treated surfaces was thenmeasured, as shown on the Y-axis in arbitrary units in FIG. 13 . Theleft bar in FIG. 13 shows proliferation on non-silver-loaded matrices,the middle bar shows proliferation on matrices that were silver-loadedby soaking in 0.001M [Ag(NH₃)₂]NO₃ for 30 minutes, and the bar on theright shows proliferation on matrices that were silver-loaded by soakingin 0.001M [Ag(NH₃)₂]NO₃ for 60 minutes. As can be seen in FIG. 13 , bonecell growth on anodized Ti-6Al-4V soaked in 0.001M solution for 30minutes was equivalent to that on non-silver-loaded surface, whileincreasing silver content on the matrix through prolonged soaking timecan reduce bone cell growth. Embodiments of the present invention allowfor the avoidance of such bone growth detriment, through controlledsustained antimicrobial agent release via the inventive multi-layeringapproach.

Example 12: Modification of Nanostructures that May be Used inEmbodiments of the Invention

FIG. 14 is an SEM of titanium alloy Ti-6Al-4V anodized, for example, in0.5% HF/16.7 H₂O/DMSO solution for 2 hours at 40 volts and treated with0.001M [Ag(NH₃)₂]NO₃ for 30 minutes. As will be readily appreciated bypersons having ordinary skill in the art, the tube size, length, andinter-tube space can be modified by using different parameters.Specifically, the anodization parameters, including voltage, currentdensity, duration, electrolyte composition, pH, and temperature, may becontrolled to customize the length, tube size, and inter-tube spacing ofthe nanotube structures which in turn influence the loading andreleasing of silver ions. For example, increasing the voltage mayincrease tube size and inter-tubular spacing and increasing voltageduration may increase tube structure length.

For example, as seen in FIG. 15 , a line graph shows the results of whenTi-6Al-4V is anodized in 16.7% water/DMSO/0.5% HF electrolyte under 40volts for 2 hours at 50 degrees C. and loaded with silver ions by singletime soaking in 0.001M [Ag(NH₃)₂]NO₃ for 30 minutes. The resultantmatrix inhibits S. aureus growth up to 8 days compared to 3 days ofTi-6Al-4V anodized in water/0.5% HF electrolyte under 10 volts for 10minutes and loaded with silver ions by single time soaking in 0.001M[Ag(NH₃)₂]NO₃ for 30 minutes. The increased inhibition period isconsidered a result of increased amount of Ag ions loaded into thenanotubes which have larger size, length and interspaces than Ti-6Al-4Vanodized in water/0.5% HF electrolyte under 10 volts for 10 minutes.

Example 13: Antimicrobial Medical Device Comprising a CeramicAntimicrobial Loaded Layer

A further embodiment of the invention includes a medical devicecomprising a ceramic layer (e.g., calcium phosphate). In someembodiments, the ceramic layer comprises atoms of an antimicrobial metalselected from the group consisting of silver, copper, and zinc, andcombinations thereof. FIG. 16 shows an SEM image of titanium alloyTi-6Al-4V, which was ultimately anodized in 0.5% HF solution for 10minutes at 10 volts, then coated with calcium phosphate using anelectro-deposition process, and finally treated with 0.001M[Ag(NH₃)₂]NO₃ for 30 minutes, thereby resulting of loading of silverions into the ceramic. In various embodiments, the first and secondantimicrobial oxide layers are formed below the ceramic layer (i.e.,between the metal surface and the ceramic layer).

FIG. 17 is a spectrum of surface chemistry by EDS which indicates theexistence of calcium phosphate and incorporation of silver ions into theceramic. In addition, the nanostructures formed during anodization ofthe Ti-6Al-4V were also all covered by the calcium phosphate.

FIG. 18 is a line graph that shows S. aureus growth in a culture wellcontaining a Ti-6Al-4V sample (control) and the silver loaded calciumphosphate coated Ti-6Al-4V disc of the present example. The Y-axis isarbitrary units. Note that the 10⁷/ml seeding density appears to be muchmore than physiological relevant value ˜10⁴/ml. The control substratehas bacteria growth from day 1 reaching a detecting limit while thesilver loaded calcium phosphate coated substrate inhibited bacteriagrowth up to 4 days.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprise” (andany form of comprise, such as “comprises” and “comprising”), “have” (andany form of have, such as “has” and “having”), “include” (and any formof include, such as “includes” and “including”), “contain” (and any formcontain, such as “contains” and “containing”), and any other grammaticalvariant thereof, are open-ended linking verbs. As a result, a method ordevice that “comprises”, “has”, “includes” or “contains” one or moresteps or elements possesses those one or more steps or elements, but isnot limited to possessing only those one or more steps or elements.Likewise, a step of a method or an element of a device that “comprises”,“has”, “includes” or “contains” one or more features possesses those oneor more features, but is not limited to possessing only those one ormore features. Furthermore, a device or structure that is configured ina certain way is configured in at least that way, but may also beconfigured in ways that are not listed.

As used herein, the terms “comprising,” “has,” “including,”“containing,” and other grammatical variants thereof encompass the terms“consisting of” and “consisting essentially of.”

The phrase “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed composition, device or method.

All publications cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

Subject matter incorporated by reference is not considered to be analternative to any claim limitations, unless otherwise explicitlyindicated.

Where one or more ranges are referred to throughout this specification,each range is intended to be a shorthand format for presentinginformation, where the range is understood to encompass each discretepoint within the range as if the same were fully set forth herein.

While several aspects and embodiments of the present invention have beendescribed and depicted herein, alternative aspects and embodiments maybe affected by those skilled in the art to accomplish the sameobjectives. Accordingly, this disclosure and the appended claims areintended to cover all such further and alternative aspects andembodiments as fall within the true spirit and scope of the invention.

The invention claimed is:
 1. A method of reducing colonization ofbacteria comprising: providing an antimicrobial medical device, thedevice comprising: a substrate comprising a metal surface, said metalsurface comprising atoms of at least one of a metal or metal alloycomprising one or more of stainless steel, cobalt, and titanium; whereinon the metal surface, a first antimicrobial oxide layer comprising atomsof the metal or metal alloy and atoms of an antimicrobial metal selectedfrom the group consisting of silver, copper, and zinc, and combinationsthereof, the atoms of the antimicrobial metal being present in the firstantimicrobial oxide layer in a first concentration; wherein on the firstantimicrobial oxide layer, positioned in a direction opposite that ofthe metal surface, a second antimicrobial oxide layer comprising atomsof an antimicrobial metal selected from the group consisting of silver,copper, and zinc, and combinations thereof, the atoms of theantimicrobial metal being present in the second antimicrobial oxidelayer in a second concentration; and wherein the first concentration isnot equal to the second concentration; and implanting the antimicrobialmedical device in a subject under conditions effective to reducecolonization of bacteria.
 2. The method according to claim 1, whereinthe oxide in the second antimicrobial oxide layer comprises silveratoms.
 3. The method according to claim 1, wherein the oxide in thesecond antimicrobial oxide layer comprises atoms of at least one ofstainless steel, cobalt, and titanium.
 4. The method according to claim1, wherein the first and second antimicrobial oxide layers are in directcontact with one another.
 5. The method according to claim 1, whereinthe first concentration is greater than the second concentration.
 6. Themethod according to claim 1, wherein the first concentration is lessthan the second concentration.
 7. The method according to claim 1,wherein the first antimicrobial oxide layer comprises silver atoms. 8.The method according to claim 1, wherein the medical device is selectedfrom an orthopedic implant and a neurosurgical implant.
 9. The methodaccording to claim 1 further comprising: promoting bone ingrowth into ascaffold.
 10. The method according to claim 1 further comprising:promoting securement of the antimicrobial medical device.
 11. The methodaccording to claim 1, wherein the metal surface is fabricated from atleast one of titanium, a titanium alloy, stainless steel, and acobalt-chrome alloy.
 12. The method according to claim 11, wherein themetal surface is selected from at least one of a Ti6Al4V and acobalt-chrome alloy.
 13. The method according to claim 1, wherein thefirst and second antimicrobial oxide layers are distinct layersseparated from one another by at least an intermediate layer that doesnot comprise an antimicrobial metal selected from the group consistingof silver, copper, and zinc, and combinations thereof.
 14. The methodaccording to claim 13, wherein the intermediate layer comprises atoms ofat least one of stainless steel, cobalt, and titanium.
 15. The methodaccording to claim 1 further comprising: providing a ceramic layer, theceramic layer comprising atoms of an antimicrobial metal selected fromthe group consisting of silver, copper, and zinc, and combinationsthereof.
 16. The method according to claim 15, wherein the ceramic layercomprises calcium phosphate.
 17. The method according to claim 1 furthercomprising: releasing one or one or more silver ions from the medicaldevice.
 18. The method according to claim 17, wherein the silver ionsbind to one or more layers of a surface oxide.
 19. The method accordingto claim 1, wherein the device further comprises a plurality ofnanostructures disposed on the metal surface, and wherein said first andsecond antimicrobial oxide layers are contained within the plurality ofnanostructures.
 20. The method according to claim 19, wherein theplurality of nanostructures are formed by at least one of anodizationand soaking.
 21. The method according to claim 19, wherein thenanostructures are at least one of nanotubes, amorphous non-crystallinenanostructures, or titanium dioxide nanostructures.